t

       *£>-•<
  ; V"
iif.trt C" total energy consumed
   P« production, both somatic and
     reproductive
   R» energy lost through hea'
     production (respiration)
   U* energy lost o* excreta
   F « energy Lost as feces

     C-F-A-P4-R+U Or

   P (Scope for Growth)-A -IR + U)
                                    FIELD VERIFICATION PROGRAM
                                         (AQUATIC DISPOSAL)

                                        TECHNICAL REPORT D-85-6

                         UTILITY OF THE SCOPE FOR  GROWTH  INDEX
                         TO  ASSESS THE PHYSIOLOGICAL IMPACT  OF
                        BLACK  ROCK  HARBOR SUSPENDED  SEDIMENT
                          ON THE  BLUE  MUSSEL,  MYTILUS EDULIS:
                                 A LABORATORY EVALUATION

                                                 by
                              William G.  Nelson, Dianne Black, Donald  Phelps
                                    Environmental Research Laboratory
                                   US Environmental Protection Agency
                                    Narragansett, Rhode Island 02882
                                        September 1985
                                         Final Report

                                Approved For Public Release; Distribution Unlimited
                             Prepared for DEPARTMENT OF THE ARMY
                                  US Army Corps of Engineers
                                  Washington, DC  20314-1000
                             and US Environmental Protection Agency
                                    Washington, DC  20460
                               Monitored by Environmental  Laboratory
                         US Army  Engineer Waterways Experiment Station
                          PO Box 631, Vicksburg, Mississippi  39180-0631

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   Destroy this report when  no longer  needed. Do not return
                      it to the originator.
The findings in this  report are not to be construed  as  an official
     Department  of the Army position unless so designated
                by other authorized documents.
       The contents of this report  are not to be used  for
        advertising, publication,  or  promotional  purposes.
         Citation of trade names does not constitute an
         official endorsement or approval of the use of
                  such  commercial products.
       The D-series of reports  includes publications of the
          Environmental  Effects of Dredging Programs:
            Dredging Operations Technical  Support
           Long-Term Effects of Dredging Operations
       Interagency Field Verification  of Methodologies  for
        Evaluating Dredged Material  Disposal Alternatives
                  (Field Verification  Program)

-------
 SUBJECT:  Transmittal of Field Verification Program Technical Report Entitled
          "Utility of the Scope for Growth Index to Assess the Physiological
          Impact of Black Rock Harbor Suspended Sediment on the Blue Mussel,
          Mytilys edulis:  A Laboratory Evaluation"

 TO:  All Report Recipients
1.  This  is one in a series of scientific reports documenting the findings of
studies conducted under the Interagency Field Verification of Testing and
Predictive Methodologies for Dredged Material Disposal Alternatives (referred
to as the Field Verification Program or FVP).  This program is a comprehensive
evaluation of environmental effects of dredged material disposal under condi-
tions of upland and aquatic disposal and wetland creation.

2.  The FVP originated out of the mutual need of both the Corps of Engineers
(Corps) and the Environmental Protection Agency (EPA) to continually improve
the technical basis for carrying out their shared regulatory missions.  The
program is an expansion of studies proposed by EPA to the US Army Engineer
Division, New England (NED), in support of its regulatory and dredging mis-
sions related to dredged material disposal into Long Island Sound.  Discus-
sions among the Corps1 Waterways Experiment Station (WES), NED, and the EPA
Environmental Research Laboratory (ERLN) in Narragansett, RI, made it clear
that a dredging project at Black Rock Harbor in Bridgeport, CT, presented a
unique opportunity for simultaneous evaluation of aquatic disposal, upland
disposal, and wetland creation using the same dredged material.  Evaluations
were to be based on technology existing within the two agencies or developed
during the six-year life of the program.

3.  The program is generic in nature and will provide techniques and inter-
pretive approaches applicable to evaluation of many dredging and disposal
operations.  Consequently, while the studies will provide detailed site-
specific information on disposal of material dredged from Black Rock Harbor,
they will also have great national significance for the Corps and EPA.

4.  The FVP is designed to meet both Agencies' needs to document the effects
of disposal under various conditions, provide verification of the predictive
accuracy of evaluative techniques now in use, and provide a basis for deter-
mining the degree to which biological response is correlated with bioaccumula-
tion of key contaminants in the species under study.  The latter is an
important aid in interpreting potential biological consequences of bioaccumu-
lation.  The program also meets EPA mission needs by providing an opportunity
to document the application of a generic predictive hazard-assessment research
strategy applicable to all wastes disposed in the aquatic environment.  There-
fore, the ERLN initiated exposure-assessment studies at the aquatic disposal
site.  The Corps-sponsored studies on environmental consequences of aquatic
disposal will provide the effects assessment necessary to complement the EPA-
sponsored exposure assessment, thereby allowing ERLN to develop and apply a
hazard-assessment strategy.  While not part of the Corps-funded FVP, the EPA
exposure assessment studies will complement the Corps'  work, and together the
Corps and the EPA studies will satisfy the needs of both agencies.

-------
SUBJECT:  Transmittal of Field Verification Program Technical Report Entitled
          "Utility of the Scope for Growth Index to Assess  the Physiological
          Impact of Black Rock Harbor Suspended Sediment  on the Blue Mussel,
          Mytilus edulis;  A Laboratory Evaluation"

5.  In recognition of the potential national significance,  the Office,  Chief
of Engineers, approved and funded the studies in January 1982.   The work is
managed through the Environmental Laboratory's Environmental Effects of
Dredging Programs at WES.  Studies of the effects of upland disposal and
wetland creation are being conducted by WES and studies of  aquatic disposal
are being carried out by the ERLN, applying techniques worked out at the
laboratory for evaluating sublethal effects of contaminants on aquatic  organ-
isms.  These studies are funded by the Corps while salary,  support facilities,
etc., are provided by EPA.  The EPA funding to support the  exposure-assessment
studies followed in 1983; the exposure-assessment studies are managed and
conducted by ERLN.

6.  The Corps and EPA are pleased at the opportunity to conduct cooperative
research and believe that the value in practical implementation and improve-
ment of environmental regulations of dredged material disposal will be  con-
siderable.  The studies conducted under this program are scientific in  nature
and will be published in the scientific literature as appropriate and in a
series of Corps technical reports.  The EPA will publish findings of the
exposure-assessment studies in the scientific literature and in EPA report
series.  The FVP will provide the scientific basis upon which regulatory
recommendations will be made and upon which changes in regulatory implementa-
tion, and perhaps regulations themselves, will be based.   However, the  docu-
ments produced by the program do not in themselves constitute regulatory
guidance from either agency.  Regulatory guidance will be provided under
separate authority after appropriate technical and administrative assessment
of the overall findings of the entire program.
      Choromokos, Jr., Ph.D., P.E.
Director, Research and Development
U. S. Army Corps of Engineers
Bernard D. Goldstein, H.D.
Assistant Administrator for
Research and Development
U. S. Environmental Protection
Agency

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      Unclassified
SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered)
REPORT DOCUMENTATION PAGE
1. REPORT NUMBER 2. GOVT ACCESSION NO.
Technical Report D-85-6
4. TITLE (md Subtitle)
UTILITY OF THE SCOPE FOR GROWTH INDEX TO ASSESS
THE PHYSIOLOGICAL IMPACT OF BLACK ROCK HARBOR
SUSPENDED SEDIMENT ON THE BLUE MUSSEL, MYTILUS
EDULIS: A LABORATORY EVALUATION
7. AUTHORO)
William G. Nelson, Dianne Black, Donald Phelps
9. PERFORMING ORGANIZATION NAME AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
11. CONTROLLING OFFICE NAME AND ADDRESS
DEPARTMENT OF THE ARMY, US Army Corps of Engineers
Washington, DC 20314-1000 and US Environmental
Protection Agency, Washington, DC 20460
14. MONITORING AGENCY NAME A ADDRESSf/f different tram Controlling Olllce)
US Army Engineer Waterways Experiment Station
Environmental Laboratory
PO Box 631, Vicksburg, Mississippi 39180-0631
READ INSTRUCTIONS
BEFORE COMPLETING FORM
3. RECIPIENT'S CATALOG NUMBER
5. TYPE OF REPORT 4 PERIOD COVERED
Final report
6. PERFORMING ORG. REPORT NUMBER
8. CONTRACT OR GRANT NUMBERf.)
10. PROGRAM ELEMENT, PROJECT, TASK
AREA & WORK UNIT NUMBERS
Field Verification Program
(Aquatic Disposal)
12. REPORT DATE
September 1985
13. NUMBER OF PAGES
56
IS. SECURITY CLASS, (ol thl* report)
Unclassified
15a. DECLASSIFI CATION/DOWNGRADING
SCHEDULE
 IS. DISTRIBUTION STATEMENT (ol thl. Report)

  Approved for public release; distribution unlimited.
 17. DISTRIBUTION STATEMENT (ol th* fbetract entered In Block 30, It dltterent (torn Report)
 IB. SUPPLEMENTARY NOTES
  Available from National Technical Information  Service, 5285 Port Royal Road,
  Springfield, Virginia  22161.
19. KEY WORDS (Continue on reran* tld» If necoieery and Identity by block number)
  Mussels—Environmental aspects   (LC)        Mytilus edulis   (WES)
  Dredging—Environmental aspects   (LC)       Dredging—Connecticut—
  Dredged material   (WES)     .                   Black Rock Harbor   (LC)
  Marine pollution   (LC)
20. ABSTRACT (Vaatlaue en nrerme eMt H meMMfjr and Identity by block number;
       The sensitivity, variability, and reproducibility of the scope for growth
  index (SFG) as  an  indicator of  physiological condition was  evaluated utilizing
  the blue mussel, Mytilus edulis,  after exposure to highly contaminated dredged
  material.  A preliminary experiment was completed to determine a no-observable-
  effects-concentration due to  suspended reference sediment (REF) alone (50 mg/Jl)
  The effect of contaminated dredged material from Black Rock Harbor (BRH) was
  then tested using  three treatments of suspended sediment:   (a) 50 mg/X, of BRH
  sediment (100 BRH), (b) 25 mg/£ each of BRH and REF sediment  (Continued)
DO/0""
     JAM 73
1473
EDITION OF I MOV 65 IS OBSOLETE
                                                       Unclassified
                                            SECURITY CLASSIFICATION Of THIS PAGE (Wrten Dele Entered)

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   Unclassified
SECURITY CLASSIFICATION OF THIS PAOE(TWl«n Dmtm Bntfnd)
 20.  ABSTRACT (Continued).

 (50-50 BRH/REF),  and (c)  50 mg/£ REF sediment alone (100 REF).  This 26-day
 bioassay  demonstrated a significant SFG reduction in mussels exposed to 100 BRH
 sediment  (-3.63 J/hr) and the 50-50 BRH/REF treatment (-2.32 J/hr) compared to
 mussels exposed to 100 REF sediment (2.53 J/hr).  This experiment was replicated
 to evaluate  the reproducibility of the technique.  The second experiment produc-
 ed similar results with the 100 REF treatment mussels having a significantly
 higher SFG (10.22 J/hr) than both the 50-50 BRH/REF (0.51 J/hr) and 100 BRH
 (-1.07 J/hr)  mussels.  The  data indicate that in a laboratory exposure the use
 of the SFG index  with M.  edulis provides a sensitive and reproducible technique
 for determining the chronic negative impact due to this dredged material.
     This investigation is  the first phase in developing field-verified bio-
 assessment evaluations for  the Corps of Engineers and the U.S. Environmental
 Protection Agency regulatory program for dredged material disposal.  This
 report is not suitable for  regulatory purposes; however, appropriate assessment
methodologies that are field verified will be available at the conclusion of
 this program.
                                             Unclassified
                                        SECURITY CLASSIFICATION OF THIS PAGEflTh.n DM* Entered)

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                               PREFACE






     This report describes work performed by the U.S. Environmental




Protection Agency (EPA), Environmental Research Laboratory,




Narragansett, R.I. (ERLN), as part of the Interagency Field Verifica-




tion of Testing and Predictive Methodologies for Dredged Material




Disposal Alternatives Program (Field Verification Program (FVP)).




The program is sponsored by the Office, Chief of Engineers (OCE),




and administered by the U.S. Army Engineer Waterways Experiment




Station (WES), Vicksburg,  Miss.,  under the purview of the Environmen-




tal Laboratory (EL).   The OCE Technical Monitors were Dr. John Hall




and Dr. William L. Klesch.  The objective of this interagency




program is to evaluate the environmental consequences of dredged




material disposal under aquatic,  wetland, and upland conditions.




The aquatic portion of the FVP study is being conducted by ERLN,




with the wetland and upland portions conducted by WES.




     The principal investigators for this aquatic study were Mr.




William Nelson, Ms. Dianne Black, and Dr. Donald Phelps, all of ERLN.




Assistance in the design and maintenance of the laboratory system was




provided by Ms. Melissa Hughes and Mr. Greg Tracey.  Laboratory-




cultured algae were also provided by Mr. Tracey.  Technical support for




the scope for growth measurements was provided by Mr. William Giles.




In addition, assistance in statistical analysis was provided by Drs.




James Heltshe and Clifford Katz.




     The EPA Technical Director for the FVP was Dr. John H. Gentile;




Technical Coordinator was Mr. Walter Galloway; and the Project Manager




was Mr. Allan Beck.






                                  1

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     The study was conducted under the direct management of

Dr. Thomas M. Dillon and Dr. Richard K. Peddicord of the Contam-

inant Mobility and Criteria Group (CMCG), Ecosystem Research and

Simulation Division (ERSD), EL; and the general management of

Dr. Charles R. Lee, Chief, CMCG, Mr. Donald L. Robey, Chief, ERSD,

and Dr. John Harrison, Chief, EL.  Mr. Charles C. Calhoun, Jr., and

Dr. Robert M. Engler were Program Mangers of the EL Environmental

Effects of Dredging Programs.

     During the preparation of this report, COL Tilford C. Creel,

CE, and COL Robert C. Lee, CE, were Commanders and Directors of

WES and Mr. F. R. Brown was Technical Director.  At the time of

publication, COL Allen F. Grum, USA, was Director and Dr. Robert W.

Whalin was Technical Director.

     This report should be cited as follows:

     Nelson, W.G., Black, D., and Phelps, D.  1985.  "The Utility
     of the Scope for Growth Index to Assess the Physiological
     Impact of Black Rock Harbor Suspended Sediment on the Blue
     Mussel, Mytilus edulis;  A Laboratory Evaluation," Technical
     Report D-85-6, prepared by US Environmental Protection
     Agency, Narragansett, R.I., for the US Army Engineer Water-
     ways Experiment Station, Vicksburg, Miss.

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                               CONTENTS
PREFACE	    1

LIST OF FIGURES	    A

LIST OF TABLES	    5

PART I: INTRODUCTION	    6

       Background.	    6
       Purpose.	    6
       Scope	    7

PART II:  MATERIALS AND METHODS	„	   10

       Overview.	   10
       Sediment Collection and Storage	   10
       Mussel Collection	   12
       Experimental Design.	-	   12
       Scope for Growth Methods	   17

PART III: RESULTS	   24

       Exposure System Monitoring	   24
       Physiological Parameters	   25
       Scope for Growth Index	   28

PART IV:  DISCUSSION	   33

PART V: CONCLUSIONS AND RECOMMENDATIONS	   38

REFERENCES	   40

APPENDIX A:  No-Observable-Effeet-Concentration Test...   Al

       Introduction	   A2
       Materials and Methods	   A2
       Results	   A4
       Discussion.	   A9

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                           LIST OF FIGURES
No.                                                         Page

1     Central Long Island Sound disposal site and
      South reference site	.,	   11

2     Black Rock Harbor, Connecticut, source of dredged
      material	   11

3     Sediment dosing system with chilled water bath
      and argon gas supply	   15

4     Exposure system used in Black Rock Harbor
      experiments.	   15

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                            LIST OF TABLES
No.                                                             Page

1    Daily monitoring data for Black Rock Harbor Experiments
     One and Two	    24

2    The mean (SE) weight-specific clearance rates of mussels
     from the two BRH experiments	    26

3    The mean (SE) absorption efficiencies of mussels exposed
     to three exposure treatments in the two BRH experiments.    26

4    Mean (SE) weight-specific respiration rates of mussels
     exposed to three types of suspended sediments	    27

5    The mean (SE) ammonia excretion rates for mussels from
     the two BRH exposure experiments	    28

6    The mean (SE) weight-specific scope for growth values
     for the three exposure treatments in the two BRH
     experiments	    29

7    The mean 0:N ratios of each treatment in Experiments
     One and Two	    30

8    The coefficients of variation (percent) for each of the
     physiological parameters, the SFG index, and the 0:N
     ratio	    31

9    Mean dry weight and length of mussels from each
     treatment in the BRH experiments	    32

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   UTILITY OF THE SCOPE FOR GROWTH INDEX TO ASSESS THE PHYSIOLOGICAL
   IMPACT OF BLACK ROCK HARBOR SUSPENDED SEDIMENT ON THE BLUE MUSSEL,
                MYTILUS EDULIS:  A LABORATORY EVALUATION
                        PART I:  INTRODUCTION


                              Background

       1.  The U.S. Army Corps of Engineers (CE) and the U.S.

Environmental Protection Agency (EPA) are jointly conducting a com-

prehensive Field Verification Program (FVP).  The approach being

used in the FVP Is to evaluate and field validate assessment method-

ologies for predicting the environmental impacts of dredged material

disposal in aquatic, upland, and wetland environments.  The research,

evaluation, and field verification of the upland and wetland disposal

options will be conducted by the Environmental Laboratory, U.S. Army

Engineer Waterways Experiment Station (WES), Vicksburg, Miss.  The

application and field verification of predictive methodologies for

the aquatic disposal option will be conducted by the EPA Environmental

Research Laboratory (ERLN), Narragansett, R.I.

                               Purpose

       2.  There are three major objectives in the aquatic portion

of the FVP at ERLN with respect to the scope for growth (SFG) index.

The first objective is to evaluate the sensitivity, variability, and

reproducibility of the SFG index.  The blue mussel, Mytilus edulis,

will be exposed to the same level of Black Rock Harbor (BRH) material in

two separate laboratory experiments.  The mussels will then be physiolog-

ically assessed using the SFG index, and the results of each experiment

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evaluated to establish the accuracy and reproducibility of the tech-




nique.  This constitutes the Laboratory Documentation Phase of the




FVP and is the subject of this report.




       3.  Subsequently, a second objective will reproduce field level




exposures in the laboratory and observe whether laboratory results




accurately predict what is observed in the field.  This is termed the




Field Verification Phase.  A third objective will determine the degree




of correlation of tissue residues resulting from the bioaccumulation




of contaminants from dredged material and ecologically significant




alterations in organism viability as observed in both the laboratory




and the field.




                                Scope




       4.  The SF6 index (Warren and Davis 1967) is a measure of the




energy available to an organism for production, both somatic and




reproductive, after routine metabolic costs are accounted for.  This




index has had extensive application with M«_ edulis ranging from the




investigation of the effects of ration levels on mussels (Thompson




and Bayne 1974) to the effects of estuarine pollution levels on




mussels (Widdows, Phelps, and Galloway 1981).  In addition, an eco-




logical relevance of the SFG index has been described by Bayne, Clark,




and Moore (1981).  They state that a sustained reduction in SFG




results in decreased growth efficiencies, subsequently smaller indi-




viduals, and ultimately reduced fecundity and fitness.




       5.  While SFG has been proven useful in other applications,




its use in this component of the FVP is to document the usefulness

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and reproducibility of the scope for growth index (SFG) as a physiolog-




ical endpoint in Mytilus edulis for measuring chronic effects of highly



contaminated dredged material.  In order to fulfill the Laboratory Docu-




mentation requirements of the FVP, the sensitivity of this technique was




tested using a contaminated sediment (BRH sediment) and a reference sedi-



ment as the exposure materials.  Reproducibility was assessed by com-



parison of the results from two separate experimental exposures.




       6.  A very important point must be emphasized at the begining



of this report.  SFG may be used to test two different hypotheses.



The first hypothesis is that differences in exposure conditions (i.e.,



levels of suspended particulates, types of sediments, etc.) have no




direct and immediate effect on the SFG index.  To test this hypothesis



the conditions under which SFG is measured replicate the experimental




exposure conditions.



       7.  The second hypothesis is that there is no chronic effect due




to differences between experimental exposures.  To test this hypothesis




the conditions under which SFG is measured are standardized and in no



way attempt to replicate the actual experimental exposure conditions.



Because standardized conditions are employed, only relative differences



between the treatments of an experiment can be compared to evaluate



chronic effects.  Comparisons of absolute SFG values between experi-



ments are not completely valid and must be made .carefully.  It is



this second application that is being evaluated in the present FVP



testing.  All statements and comparisons concerning SFG effects and



reproducibility must be considered with this fact in mind.
                                   8

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       8.  This report details two experiments.   The first experiment




establishes the effect of a 50-mg/Jl suspended sediment exposure




of:  (a) 100 percent BRH sediment (100 BRH),  (b) 100 percent reference




sediment (100 REF), and (c) a 50 percent-50 percent mix of each




sediment (50-50 BRH/REF) on the SFG of M^ edulis.  The second exper-




iment, a replicate of the first, documents the reproducibility of




the results obtained.  In addition, a preliminary experiment (Appendix




A) was completed to establish a "no-observable-effect-concentration"




(NOEC) of suspended reference sediment with respect to the SFG index.




The laboratory exposure levels were selected strictly as the NOEC.




There was no expectation that these levels were in any way represent-




ative of suspended sedimentary levels actually occurring in central




Long Island Sound.

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                   PART  II:  MATERIALS AND METHODS





                               Overview



       9.  The tests described below generally follow methods pre-



scribed in "Standard Practice for Conducting Acute Toxicity Tests



with Fishes, Macroinvertebrates, and Amphibians" (ASTM 1980).  Although



the ASTM test methods were not specifically designed for sediment



tests, they provide guidelines for experimental designs, water quality



parameters, statistical analyses, and animal care, handling, and



acclimation.






                   Sediment Collection and Storage



       10.  Two sediment types were used to conduct the suspended



particulate tests in this study.  The reference sediment (REF) was



collected from the South reference site in Long Island Sound (4l°7.95"N



and 72C52.7"W) by a Sraith-Maclntyre grab (0.2 m ),  press sieved through



a 2-mm sieve, and stored at 4°C until used (Figure 1).  Black Rock



Harbor (BRH) sediment was collected from the highly contaminated and



industrallzed dredge site (41°9"N and 73°13"W) with a gravity box corer


      2
(0.1 m ) to a depth of 1.21 m, thoroughly mixed, press sieved through a



2-mm sieve, and refrigerated (4°C) until used (Figure 2).  In all



experiments, sediments were allowed to reach test temperature and mixed



prior to use.
                                  10

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                                                     SOUTH REFERENCE
                                                          • SITE
       Figure  1.   Central Long Island Sound disposal  site  and
                  South reference site.
                                       MAINTENANCE
                                       DREDGING
Figure 2.
Black Rock Harbor, Connecticut,  source of dredged material,

                       11

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                          Mussel Collection

       11.  Mussels for each experiment, the preliminary one (NOEC)

and the two BRH exposures (experiments one and two-, were collected

in a similar manner with a scallop dredge from an uncontaminated

site near Dutch Island in the west passage of Narragansett Bay

(71°24.0'W by 41°29.4'N) from depths ranging between 5 and 10 m.

Collection information for each experiment is listed below:
             Collection       Experiment      Field          Field
Experiment      Date            Begun      Temperature °C  Salinity °/oo
NOEC 10/7/83
Experiment 1 11/10/83
Experiment 2 3/8/84
10/11/83
11/16/83
3/19/84
17.5
13.0
5.0
31.0
31.0
29.0
The animals were sorted to obtain a size range of 50 to 55 mm shell

length and held in a laboratory flow-through system at ambient

temperature and in unfiltered seawater until the experiment was

initiated.  All experiments were run at 15°C.  Mussels collected

from the field when the temperature was below 15°C were acclimated

in running unfiltered seawater at a rate of 1°C per day until 15°C

was reached.


                         Experimental Design


No-observable-effeet-concentration experiment

       12.  In order to obtain an effect with BRH material it was

believed that the mussels should be exposed to the highest reasonable

level of suspended particulates.  The determination of a reasonable
                                  12

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particulate level was termed the no-observable-effeet-concentration




(NOEC) experiment.  This experiment is detailed in Appendix A.




Briefly, two criteria were used in selecting the NOEC:   (a) maximum




exposure to the. suspended sediment, and (b) no significant reduction




in SFG.  The approach taken in the NOEC experiment was  to expose




mussels to different particulate concentrations (0, 6.25,  12.5,  25,




50, and 100 mg/£) of suspended reference sediment.   The results




indicated that mussels in the 50-mg/£ treatment produced pseudofeces




throughout the 28-day experiment, while those in the lower treatment




levels did not.  This would maximize processing of, and thus exposure




to, the suspended material through the gastrointestinal tract of




the animal.  In addition, mussels from this treatment also exhibited




the highest SFG values.  For these reasons, a NOEC level of 50 mg/A




was selected.






Experiments one and two




       13.  The first experiment in the laboratory documentation




phase of the FVP was designed to establish the sensitivity of the




SFG index as a measure of impact, in this case due to possible effects




of BRH dredged material on M^ edulis.  This experiment  was repeated




a second time to further document the observed sensitivity and thus




determine the degree of reproducibility.  The following methodology




applies to both BRH experiments.
                                   13

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Exposure system




       14.  Implementation of the experimental design required the




construction of two identical sediment dosing systems to simultaneously




provide either BRH or REF material as suspended sediment.  The dosing




system (Figure 3) consisted of conical-shaped slurry reservoirs placed




in a chilled fiberglass chamber, a diaphram pump, a 4-A separatory




funnel, and several return loops that directed the particulate slurry




through the dosing valves.  The slurry reservoirs (40 cm diam.  x 55 cm




high) contained 40 H of slurry composed of 37.7 A of filtered seawater




and 2.3 A of either BRH or REF sediment.  The fiberglass chamber (94 cm




x 61 cm x 79 cm high) was maintained between 4° and 10°C using an




externally chilled water source.  (The slurry was chilled to minimize




microbial degradation during the test.) Polypropylene pipes (3.8 cm




diam.) placed at the bottom of the reservoir cones were connected to




the diaphragm pumps (16 to 40 i/min capacity) that had Teflon




diaphragms.  These pumps were used to circulate the slurry but minimize




abrasion so that the physical properties and particle sizes of the




material remained as unchanged as possible.
                                  14

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                   SEPARATORY
                    FUNNEL
                                          DELIVERY
                                          MANIFOLD
                                                   DOSING
                                                   VALVE
                                               TO EXPOSURE
                                                SYSTEM
                                            SLURRY
                                            RESERVOIR
                Figure  3.   Sediment dosing system
                              with chilled water bath
                              and  argon  gas  supply.
        reference slurry
L
                             MUSSEL EXPOSURE  SYSTEM
                seowoler
                                                        BRH slurry
  mixing 	
   chamber
     magnetic/
      stirrer
              distribution
               chamber —
                                 siphons
                      lo drain
                                n
                                                               leawater
                                           overflow to water both, then to drain
                                            exposure chamber
                          lo drain
                                     magnetic slirrer
Figure 4.   Exposure  system  used in the Black Rock Harbor
              experiments.
                                    15

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       15.  The separatory funnel was connected to the pump and



returned to the reservoir by polypropylene pipes.  The separatory



funnel served two functions:  (a) to ensure that a .constant head




pressure was provided by the overflow, and (b) to serve as a connec-



tion for the manifold located 4 cm below the constant head level.



The manifold served to distribute the slurry by directing a portion



of the flow from the funnel (through 6 mm Inside diam. polypropylene



tubes) through the Teflon dosing valves (Figure 3) and back to the



reservoir.  At the dosing valves, the slurry was mixed with seawater



to provide the desired concentrations for the toxicity tests.  Argon



gas was provided at the rate of 200 ml/min to the reservoir and the



separatory funnel to minimize oxidation of the sediment/seawater




slurry.  Narragansett Bay seawater filtered (to 15 ym) through



sand filters was used for these experiments.  The dosing valves were



controlled by a microprocessor which was programmed to deliver a




pulse with a duration of 0.1 sec up to continuous pulse delivery and




at intervals from once every second to once every hour.



       16.  The exposure system is shown in Figure 4.  In these



experiments the REF and BRH mixing and distribution chambers (Figure 4)



were maintained at 50 mg/£.  Exposure conditions were obtained by



siphoning suspended sediment from the appropriate distribution chambers



to produce a combined flow of 300 ml/min in each exposure chamber.  The



amount of suspended particulates both entering the exposure chambers



(actual incoming concentration) and the concentration surrounding the




mussels (actual surrounding concentration) were measured daily using a
                                  16

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spectrophotometer.  Prior to the experiment,  the relationship between




absorbance and dry weight of suspended participates had been determined




by collecting triplicate samples of suspended sediment directly from




the dlluter or by preparing dilutions from the highest concentration.




The dry weight of these samples was measured using the methods reported




in Lake et al.  (1984) and their absorbance was measured on the




spectrophotometer.  Linear regression analysis of the data established




the relationship between absorbance and dry weight.  Analysis of




variance and multiple comparison tests were performed on the suspended




particulate data collected daily during the experiment.




       17.  The three exposure treatments consisted of an incoming




concentration of 50 mg/& of:  (a) 100 percent BRH sediment (100 BRH),




(b) 100 percent REF sediment (100 REF), and (c) a 50 percent-50 percent




mixture of each sediment (50-50 BRH/REF).  Forty mussels were exposed




in each treatment and fed Isochrysis aff. galbana (T-Iso) at a rate of




94 mg/mussel/day.  On day 26, ten mussels from each treatment were




sampled for SFG measurements.  The remaining mussels were distributed




for other end-point determinations.  The first experiment was




terminated after  26 days because mortality began to occur in the 100




BRH treatment.  Experiment  two was stopped after 26 days to replicate




Experiment one.






                        Scope for Growth Methods




        18.  Calculation of  the SFG index for M^ edulis required the




measurement of  four parameters:  clearance rate, respiration rate, food




absorption efficiency,  and  ammonia excretion rate.  Because the null
                                   17

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hypothesis being tested was that there would be no chronic effect due

to exposure to BRH material, the SFG measurements were completed under

standardized conditions.  All SFG measurements for a given treatment

were completed in the order shown below within 28 hrs after termination

of the experiment and all measurements were performed at 15°C.


Clearance rate

       19.  Clearance rate is defined as the volume of water completely

cleared of particles >3 microns (ym) in some unit time (Widdows et al.

1979).  In the present experiment this was measured by placing mussels

into individual chambers through which 1 ym filtered seawater flowed at

a rate of 75 ml/min.  The unicellular algae, T-Iso, was added to the

filtered seawater to deliver an incoming cell concentration of approx-

imately 25,000 cells/ml (about 0.5 mg/&) to each chamber.  Each

chamber was gently aerated to ensure that complete mixing and no

settling of algae occurred.  Mussels were allowed to acclimate in the

chambers for at least 1 hr prior to any measurements.  The incoming

and outgoing particle concentrations for each chamber were then

measured with a Coulter Counter (Model TAII) and substituted into

the following formula to determine clearance rate:


             Clearance rate - [(Cl - C2)/C2] X F      (1)
            where
                Cl and C2 - incoming and outgoing particle
                            concentrations, respectively

                        F = flow rate in liters/hour through the
                            chamber.
                                  18

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Respiration rate

     20.  Respiration rates were measured by isolating each mussel in a

glass respirometer vessel fitted with an electrode designed to measure

the partial pressure of oxygen (P02).  The electrode was connected to a

Radiometer oxygen meter (Model PHM71) which was in turn connected to a

strip chart recorder.  Each mussel was allowed to acclimate for about

10 minutes in the vessel prior to respiration measurements.  This short

acclimation period was found to be adequate by measuring the

respiration rate of several mussels for 1 hr from time of initial

placement into the vessel.  There was no change in rate after the first

5 minutes.  Seawater containing algae was pumped into the vessel during

this acclimation period at a rate of 80 ml/min to ensure that food was

present in the chamber and that routine metabolic rate was measured.

After acclimation the flow of seawater was stopped and the decline in

P02 was recorded on the strip chart recorder for approximately 30 min.

The respiration rates were calculated using the following formula:

               MMHG            RESVOL - MUSVOL      60
     ML02HR-  	X SAT02 X	X	     (2)
                160                 1000          02TIME

      where

       ML02HR « oxygen consumed per hour by the mussel, ml

         MMHG - change in partial pressure of 02 over time, mm mercury

        SAT02 - oxygen saturation level of seawater at that
                temperature, ml/A

       RESVOL - respiration vessel volume, ml

       MUSVOL - volume of the mussel, ml

       02TIME « time period of the measurement, min
                                   19

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Absorption efficiency

       21.  After completion of the respiration rate measurements, all

fecal material was removed from each feeding chamber.  This ensured

that only the algae consumed during the SFG procedures were used in the

absorption efficiency measurements.  At the food concentration used in

the SFG measurements, approximately 0.5 mg/&, no pseudofecal production

occurred.  The mussels were allowed to feed overnight in the chambers.

Fecal pellets were collected from each chamber with a Pasteur pipette

and filtered onto a 1 ym Nuclepore polycarbonate filter.  The filter

was removed to a watch glass where a few drops of isotonic ammonium

formate were added to facilitate removal of the fecal pellets.  The

fecal pellets were scraped off with a plastic spatula, deposited onto

small aluminum pans (1 cm square), and placed in a drying oven at 100°C

for 24 hr.  Pellets and pans were weighed using a Perkln Elmer

autobalance (Model AD-2Z).  Pellets were ashed at 500°C for 4 hr, and

reweighed to determine the ash-free dry weight:dry weight ratio for

the feces.  A similar procedure was completed with the cultured algae

to obtain the ash-free dry weighttdry weight ratio of the food.

Absorption efficiencies were calculated for each mussel according to

the method of Conover (1966) using the following formula:

                                    F - E
          Absorption Efficiency =	   X  100    (3)
                                  (1-E) X F

     where

          F = ash-free dry weighttdry weight ratio of the food

          E » ash-free dry weight:dry weight ratio of the feces
                                  20

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       22.  This technique allowed thb calculation of an absorption



efficiency for each mussel.  In the past, at this laboratory and in



the literature, fecal material was collected on pre-ashed glass




fiber filters which weighed a great deal more than the dried and



ashed fecal material.  The great differential between the weight of



the glass fiber filter and the weight of the dried and ashed fecal



material appeared to introduce an artifact into the data.  The sub-




stitution of the lightweight aluminum pans resulted in a 50 percent



reduction in fecal weight variability and the subsequent absorption




efficiency differences between individual mussels.






Ammonia excretion rate



       23.  Mussels were placed individually into HCl-stripped beakers



containing 300 ml of 1 ym filtered seawater for a period of 3 hr.



Mussels were then removed and a 0.45-ym filtered, 50-ml sample was




collected from each beaker, deposited into acid-stripped polyethylene



bottles, and stored in a freezer at -20°C until analyzed.  Ammonia




analyses were completed in duplicate for each sample according to the



method of Bower and Holm-Hansen (1980).






Scope for growth calculations




       24.  After completion of the physiological measurements, the



length and volume of each mussel were measured and the tissue excised,



dried for 24 hr at  100°C, and weighed.  The clearance rates, respira-



tion rates, and ammonia excretion rates were standardized to a  1 g



animal by converting the rates and dry weights to log 10, and fitting
                                  21

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the data to the allometric equation to obtain the fitted parameters, a

and b, as described by Bayne et al. (1981).  The weight-specific rates

for each mussel were determined as follows:


                                  BUte
        Weight-Specific Rate =	     (4)
                                        b
                                 Weight

where b = slope in the allometric equations calculated above.

Absorption efficiencies were found to be independent of size (slope=0)

over this narrow size range so absolute values were used.

       25.  The weight specific values for each mussel were then used to

calculate the SF6 of each individual by substitution into the following

equation:


             Scope for Growth = (C X A) - (R + E)     (5)
      where

           C = energy consumed (clearance rate X surrounding food
               concentration X energy of food)

           A - absorption efficiency

     R and E = energy lost through respiration and nitrogen excretion,
               respectively

The following energy conversions were used to calculate SFG:

           One mg of T-Iso = 4.5 X 107
           cells (this experiment)
           One mg of T-Iso = 19.24 J (this experiment)
           One ml 02 respired - 20.08 J (Crisp 1971)
           One mg NH4-N = 24.56 J (Elliot and Davidson 1975)

The energy content of T-Iso was determined by filtering a volume of the

algae onto preweighed glass fiber filters, drying them at 100°C for 24

hr, and reweighing them to determine algal dry weight.  They were then
                                  22

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analyzed using the dichromate wet oxidation method of Maciolek (1962)




to determine oxygen consumed and the resultant energy content.




     26.  Another index, the oxygen:nitrogen (0:N) ratio (the atomic




ratio of oxygen consumed to ammonia-nitrogen excreted) can also be




derived from the data obtained for the SFG calculations.  This index




was calculated for comparison with the SFG index.






Statistical analysis




     27.  Differences in physiological data and the resultant SFG




values between each treatment in the NOEC and BRH exposure experiments




were tested using one-way analysis of variance (Snedecor and Cochran




1978).  All statistical tests were completed at the 0.05 level of




significance.  Tukey's studentized range test was applied to determine




between-treatment differences.  Comparison of results between




laboratory experiments were completed using the Student's t-test, also




at the 0.05 significance level.  This distinction was made because




laboratory exposure experiments were completed at different points in




time.
                                  23

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                          PART III: RESULTS



                     Exposure System Monitoring



       28.  Data from the daily monitoring of exposure conditions for

BRH sediment experiments one and two are presented in Table 1.

                               Table 1
              Daily monitoring data for Black Rock Harbor
                       Experiments One and Two.*


                             Treatment

                     100 REF      50-50 BRH/REF     100 BRH
                          Experiment One
Actual
incoming           56.2 (8.2)       59.4 (5.5)     62.8 (9.9)
concentration
(mg particulates/fc)

Actual
surrounding        11.6 (5.1)       24.5(15.A)     30.2(17.5)
concentration
(mg particulates/£)

                          Experiment Two
Actual
incoming           49.4 (6.1)       52.9 (5.7)     56.2 (8.6)
concentration
(mg particulates/A)

Actual
surrounding        14.1 (6.4)       23.5(10.1)     29.0(11.4)
concentration
(mg particulates/£>

* Values are means with standard deviation in parentheses.
                                 24

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       29.  The data presented in Table 1  indicate that comparable




levels of sediments were delivered to each treatment,  and that  those




levels were reasonably consistent throughout the course of the  exper-




iments .






                      Physiological Parameters




       30.  The physiological results of the two experiments are




summarized in Tables 2 through 9.  In the first experiment, two mussels




died during the SFG measurements, one from each of 100 REF and  50-50




BRH/REF treatments.  As a result the mean values from these treatments




included only nine individuals, while the 100 BRH treatment means




represent ten mussels.  No mortality occurred in the second experiment;




thus all mean values included ten individuals.




       31.  The weight-specific clearance rates are listed in Table 2.




In the first experiment, the 100 REF and 50-50 BRH/REF treatments were




similar while those mussels exposed to 100 BRH were significantly




lower.  The results of the second BRH experiment indicate that the




mussels from the 100 REF treatment exhibited a significantly higher




clearance rate than the mussels from either of the other two treatments.
                                  25

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                                Table 2
       The mean (SE) weight-specific clearance rates of mussels
                     from the two BRH experiments.

          Treatment          Clearance Rate            Group*
                                 tt/hr)

100 REF
50-50 BRH/REF
100 BRH

100 REF
50-50 BRH/REF
100 BRH
Experiment One
3.81(0.23)
3.59(0.35)
2.25(0.44)
Experiment Two
3.99(0.37)
1.54(0.26)
1.40(0.34)

A
A
B

A
B
B
* Means with the same group letter are not significantly different.

       32.  The absorption efficiencies (Table 3) were all relatively

high in the first experiment with the 100 BRH and 100 REF treatments

significantly higher than the 50-50 BRH/REF group.  In the second

experiment the absorption efficiencies were even higher; however, there

were no differences between any of the treatments.

                                Table 3
           The mean (SE) absorption efficiencies of mussels
exposed to three exposure treatments in the two BRH experiments.

Treatment
100 BRH
100 REF
50-50 BRH/REF
100 REF
50-50 BRH/REF
100 BRH
Absorption Efficiency
(percent )
Experiment One
92(0.9)
89(0.8)
82(2.1)
Experiment Two
96(0.3)
96(0.3)
96(0.3)
Group*
A
A
B
A
A
A
* Means with the same group letter are not significantly different,
                                   26

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       33.  The respiration rates for each treatment  are listed in

Table 4.  There were no significant differences  between treatments

in either experiment.  The actual rates were lower in the second

experiment than in the first possibly due to seasonal differences.


                                Table 4
            Mean (SE) weight-specific respiration rates of
mussels exposed

Treatment

50-50 BRH/REF
100 REF
100 BRH

100 REF
100 BRH
50-50 BRH/REF
to three types of suspended

Respiration Rate
(ml-02/hr)
Experiment One
1.00(0.02)
0.91(0.04)
0.87(0.06)
Experiment Two
0.61(0.03)
0.53(0.04)
0.51(0.03)
sediments .

Group*

A
A
A

A
A
A
* Means with the same group letter are not significantly different.


       34.  The data listed in Table 5 indicate that the ammonia

excretion rates of the mussels from the 50-50 BRH/REF treatment were

significantly elevated as compared with those from the 100 REF group in

the  first experiment, and the same differences were evident in the

second experiment.
                                  27

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                                Table 5
               The mean (SE) ammonia excretion rates for
            mussels from the two BRH exposure experiments.
         Treatment        Ammonia Excretion Rate          Group*
                                 NH4-N/hr/g)

50-50 BRH/REF
100 BRH
100 REF

50-50 BRH/REF
100 BRH
100 REF
Experiment One
37.11(3.14)
26.88(2.81)
20.50(3.93)
Experiment Two
20.93(1.09)
19.42(2.48)
14.13(1.47)

A
A B
B

A
A B
B
* Means with the same group letter are not significantly different.


                       Scope for Growth Index


       35.  While the inter-experimental comparisons of the individual

physiological parameters are important, it is the comparison of the

integrative SFG index that is of prime interest.  The weight-specific

SFG values for each treatment are listed in Table 6.  In Experiment

One, the 100 REF group exhibited a significantly higher SFG than those

mussels exposed to 100 BRH and the 50-50 BRH/REF treatment.  The same

relative treatment differences were observed in the second BRH exper-

iment, indicating the reproducibility of the technique.  The actual

mean SFG value of the 100 REF treatment in the second experiment

(10.22 J/hr) was significantly higher than the first (2.53 J/hr),

possibly due to seasonal differences; however, the relative differences

were the same in both experiments.  This will be further detailed in

the discussion section.
                                 28

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                                Table  6
         The mean (SE)  weight-specific scope  for growth values
for the three exposure treatments in the two
BRH experiments.

Treatment
100 REF
50-50 BRH/REF
100 BRH
100 REF
50-50 BRH/REF
100 BRH
Scope for Growth
(J/hr/g)
Experiment One
2.53(0.78)
-2.32(1.17)
-3.63(1.58)
Experiment Two
10.22(1.71)
0.51(2.01)
-1.07(1.80)
Group*
A
B
B
A
B
B
* Means with the same group letter are not significantly different.

       36.  Another index,  the 0:N ratio,  was  calculated using the

respiration rate and ammonia-nitrogen excretion rate data.   The

results, Table 7, indicate  that there were no  differences in these

values between treatments in the first experiment.   In the second

experiment, the mean 0:N ratio of the 100 REF  mussels was significantly

higher than 100 BRH mussels.  The differences  and inconsistencies

between the results of this index and the SFG  index will be discussed

later in the report.
                                 29

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                                Table 7
 The mean  0;N  ratios of  each  treatment  in Experiments One and Two.

Treatment

100 REF
50-50 BRH/REF
100 BRH

100 REF
50-50 BRH/REF
100 BRH

0:N ratio
Experiment One
77
65
50
Experiment Two
63
49
34

Group*

A
A
A

A
A B
B
* Means with the same group letter are not significantly different.

       37.  One objective of the laboratory documentation phase of the

FVP is to investigate the variability of the SFG index.  One way to do

this is to look at the coefficient of variation (CV) which is the

standard deviation divided by the mean.  The absolute value of the CV

has been calculated for each physiological parameter and the SFG index

and is presented in Table 8.
                                  30

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                                Table  8
        The coefficients of  variation  (percent)  for  each  of  the
physiological parameters , the
Parameter
100
Experiment
Clearance rate
Absorption efficiency
Respiration rate
Ammonia excretion rate
Scope for growth
0:N ratio
Experiment
Clearance rate
Absorption efficiency
Respiration rate
Ammonia excretion rate
Scope for growth
0:N ratio
SFG

REF
One
18
3
14
58
92
51
Two
29
1
18
33
53
36
index, and the 0:N
Treatment
50-50 BRH/REF

29
8
7
25
152
43

53
1
18
16
1250
22
ratio.

100 BRH

63
3
21
33
138
25

76
1
24
40
530
47
       38.  The data in Table 8 would indicate that exposure to BRH

material causes a great increase in the variability associated with the

clearance rate measurement and the SFG index in both experiments.

       39.  The means of the dry weights and lengths of the mussels from

each treatment are listed in Table 9.  The results from Experiment One

indicate that the lengths of the mussels from each treatment were not

significantly different, with coefficients of variation, CV, of 2-3

percent.  The dry weights of those same animals indicated that the

mussels from the 100 REF treatment were significantly higher than the

other two treatments, with the CV increasing directly with increased

exposure to BRH material.  The results of the second experiment also

indicated that the lengths of the mussels used were very similar.
                                  31

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 Unlike the first experiment, however, there were no differences between

 the dry weights of the animals from each treatment.  There was no

 evidence of spawning in any of the treatments.
                                 Table 9
                  Mean dry weight and length of mussels
from each treatment in the
BRH experiments.
Treatment
Dry Weight
(g)
Group* CV**
f tjf V
\/o J
Experiment
100
50-50
100
REF
BRH/REF
BRH
0
0
0
.86(0
.59(0
.59(0
.04)
.04)
.06)
A
B
B
Experiment
100
50-50
100
REF
BRH/REF
BRH
0
0
0
.91(0
.88(0
.73(0
.05)
.04)
.05)
A
A
A
One
15
21
32
Two
24
15
24

5
5
5
5
5
5
Length
(cm)

.32(0.
.32(0.
.41(0.
.45(0.
.46(0.
.40(0.

05)
04)
05)
04)
01)
01)
Group*

A
A
A
A
A
A
CV
3
2
3
4
2
2
 * Means with the same group letter are not significantly different,
** CV - coefficient of variation.
                                   32

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                         PART IV:   DISCUSSION







       40.  The objective of the studies reported here is to evaluate




the sensitivity, variability, and reproducibility of the SFG index as




a method to assess the effects of BRH dredged material on M. edulis




in a laboratory exposure.  As shown in Table 6, a significant reduction




in the SFG index was observed in mussels exposed to BRH dredged material




when compared to mussels exposed to REF sediment, thus demonstrating




the sensitivity of this index.



       41.  The BRH dredged material contains polychlorinated biphenyls




(PCB) (6800 ng/g), polynuclear hydrocarbons (PAH) (9800 ng/g), and




trace metals (Cu, Cr, and Pb, at 2380, 1430, and 380 ug/g, respectively)




(Lake et al. 1984).  In the same paper M. edulis was reported to




accumulate 44% of the sediment PCB's, 28% of the sediment polynuclear




hydrocarbons, and various amounts of the Cu, Cr, and Pb trace metals




during a 28-day exposure to the BRH material.  It is likely that




the same materials were accumulated by the mussels during this experi-




ment.  A correlation is indicated between the significant differences




in the physiological parameters and SFG values and the contaminants




in the BRH material.




       42.  The data in Experiment One indicated that the mussels  from




the 100 BRH treatment exhibited1 a significantly lower clearance rate




when compared  to  the other  two treatments  (Table 2).  Stickle et al.




 (1983) reported a decreased clearance rate  in M^ edulis after a 28-




day exposure to the water-soluble fraction  of  crude oil.  Abel  (1976)
                                  33

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investigated the effects of several metals on filtration rates in




Mytilus and found that Cu, Zn, and Hg caused a reduction in this




physiological parameter.  Gilfillan et al. (1976) reported a reduced




carbon flux in the soft-shelled clam, Mya arenaria, from areas that




had been exposed to an oil spill.  They attributed this to decreased




filtration rates and higher respiration rates.  Gilfillan (1975)




showed that exposure to crude oil extracts also caused a reduction




in filtration rates in M. edulis.  Thus, sufficient evidence exists




to support the hypothesis that individual exposures to heavy metals




or oils can cause reduced filtration rates.




       43.  The results of the second experiment were similar with




differences again between the clearance rates in the 100 REF and 100




BRH treatments.  However, in the second experiment the mussels from the




50-50 BRH/REF treatment also exhibited a significantly reduced clear-




ance rate from those mussels in the 100 REF treatment.  While the




reason for this is not immediately obvious, one possible explanation




may be forwarded.  The concentration of BRH material in the 50-50




BRH/REF treatment, 25 mg/£ of BRH material, may be near the threshold




level that produces this effect.  If the mussels filtered slightly




more in the second experiment than the first, their exposure to the




the BRH material would have been increased.  A significant decrease




in the clearance rates of the 100 BRH mussels was observed in both




experiments.  Therefore, a slight difference in exposure level




(i.e., clearance rate) in the 50-50 BRH/REF treatment may have pro-




duced the reduced clearance rate in the second experiment.
                                  34

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       44.  In addition to affecting the absolute clearance rate,  BRH




dredged material also resulted in an increase in the variability of




the clearance rate measurement (Table 8).  In both experiments the




response was consistent:  an increased exposure to BRH material




resulted in a corresponding increase in the CV of this measurement.




       45.  In contrast to the lower clearance rates, no differences




were observed in respiration rates between the treatments in either




BRH experiment.   Stickle et al. (1983) found no differences in respira-




tion rates between treatments in the exposures to crude oil previously




mentioned.  During acute tests, Brown and Newell (1972) observed a




decrease in whole animal respiration rates in M^ edulis during expo-




sure to Cu.  Scott and Major (1972) reported a similar decrease in




the same species.  In contrast, Gilfillan et al. (1976) stated that




reduced carbon flow in M. arenaria was partially due to Increased




respiration rates.  Engel and Fowler (1979) found that Cu caused an




increase in respiration rate in excised oyster gill tissue.  It is




evident from the literature that no one respiration rate response  is




elicited consistently after exposure to a pollutant.  While no




differences in respiration rates were evident in the present study,




it is  important, for the purposes of this report, to note that the




same response was obtained in both experiments.



       46.  The absorption efficiencies in both experiments were quite




high.  In  the first  experiment the 50-50 BRH/REF treatment was signif-




icantly lower than the  100 REF and 100 BRH treatments  (Table 3),




while  in the second  experiment there were no differences between any




treatment.  The algae used in this study were T. Iso, a naked flagellate.

-------
This fact, along with the low food concentrations, 0.5 mg/S, used




during the SFG measurements, can account for these high efficiencies.




Widdows, Phelps, and Galloway (1981) fed Tetraselrois suecica to mussels




at a concentration of 0.35 rng/d and found similarly high absorption




efficiencies of 93 percent.  Other reported absorption efficiencies




that use such species as the diatom Phaeodactylum tricornutum or




natural seston would be expected to be lower than those reported




here.




       47.  While each individual physiological parameter measured is of




interest, the advantage of the SFG index is that it integrates the




whole animal response of each individual.  In the first experiment, the




SFG data indicate that the mussels exposed to the 50-50 BRH/REF and 100




BRH treatments exhibited significantly lower SFG values, -2.32 and




-3.63 J/hr, respectively, than those mussels from the 100 REF treatment




(2.53 J/hr).  The results of the second BRH experiment were the same as




the first one, demonstrating the reproducibility of the technique.




Those mussels from the 100 REF treatment displayed a significantly




higher SFG (10.22 J/hr) than mussels from the 50-50 BRH/REF (0.51 J/hr)




or 100 BRH (-1.07 J/hr) treatments.  The SFG results are typical of a




dose-response effect.  A similar decrease in SFG was reported for M._




edulls by Stickle et al. (1983) and for M^ arenarla by Gilfillan et al.




(1976) following exposure to oil.  Widdows et al. (1981) have also




reported a dose response type effect between SFG and aromatic petroleum




hydrocarbon exposure concentrations in M. edulls.
                                 36

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       48.  The results of another index,  0:N ratio,  are listed in




Table 7.  With this index, a lower 0:N ratio is indicative  of  a more




stressed condition.  In Experiment One there were no  differences




between treatments using this index.  The  results of  the second




experiment, however, indicated that the 100 REF treatment mussels




exhibited a significantly higher 0:N ratio than those mussels  from




the 100 BRH treatment.  The results of this index are inconsistent




with respect to the SFG index and they are also different between




the two experiments.  In addition, the 0:N ratio results do not




follow the dry weight data listed in Table 9.  The dry weight data




from Experiment One indicated that the 100 REF treatment mussels




weighed significantly more than the mussels from the other two treat-




ments.  This is the same pattern that the SFG index showed.  In the




second experiment, there were no significant differences between




treatments; however,  the order was the same as in the first experiment,




as were the SFG results.  The dry weight measurements listed in




Table  9 were made  initially  to calculate weight-specific physiological




rates, not to  infer changes  due to treatment differences.  One may




infer,  however, that  an index used  to measure  stress, such as  SFG




and  0:N ratio, might  parallel changes in actual  tissue  weight.   In




these  experiments  the SFG index corresponds more closely to these




changes  than  do the 0:N ratio.
                                   37

-------
                PART V: CONCLUSIONS AND RECOMMENDATIONS






       49.  The major objectives of the laboratory documentation portion




of the Field Verification Program were to assess the sensitivity,




reproducibility, and variability associated with various biological




indices using BRH dredged material.  The results of this experiment




indicate that the Scope For Growth index, with M^ edulis as the test




organism, is a sensitive enough technique for measuring the chronic




effects of this dredged material.




       50.  The primary purpose of this report, however, was to docu-




ment the reproducibility of this index.  The data in Table 6 indicate




that the results of one experiment can be reproduced in a second




replicate study.  The SFG value of the 100 REF treatment from experi-




ment two (10.22 J/hr) was significantly higher than the 100 REF SFG




value from the first experiment (2.53 J/hr).  This is not unexpected




because SFG values do change normally during the year with changes




in the reproductive cycle (Bayne et al. 1976).  The important point




is that the SFG values indicated the same relative response in both




BRH experiments.  This was the hypothesis being tested in this exper-




iment and the rationale for using standardized conditions in the SFG




measurements.  The results of this experiment indicate that the SFG




index, when used with M. edulis, satisfies the question of repro-




ducibility for the Laboratory Documentation phase of the FVP.




       51.  In addition to measuring the sensitivity and reproduc-




ibility of the SFG index, the effect this material has on the varia-




bility of the measurements was also of interest.  The data presented in
                                 38

-------
Table 8 would indicate that this material increases the variability



observed in several of the measurements.  The effect of BRH material




on the variability of the clearance rates has already been mentioned.




Clearance rate differences have a dramatic effect on the total amount




of energy consumed, and, therefore, on the subsequent calculation of




the SFG index.  This can also be seen from Table 8, where large



variation was also observed in the SFG index.  The exact nature of




how BRH causes more variability in this measurement is not clear.




However, it does point out one advantage of using an index such as




SFG; the physiological system that is negatively impacted may be




isolated.  In these two experiments it was apparent that BRH material




had an effect on clearance rate.  The actual mechanistic basis for




this effect can now be studied further.
                                  39

-------
                              REFERENCES
Abel, P.D.  1976.  "Effect of Some Pollutants on the Filtration Rate of
Mytilus," Marine Pollution Bulletin, vol 7, pp 228-231.

American Society for Testing and Materials, 1980.  "Standard Practice for
Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and
Amphibians," ASTM E-729-80, Philadelphia, Pa.

Bayne, B.L.  1976.  "Aspects of Reproduction in Bivalve Molluscs,"
Estuarine Processes.  Vol. 1;  Uses, Stresses and Adaption to the
Estuary, Edited by Wiley.  Academic Press, London, pp 432-448.

Bayne, B.L., Clark, K.R., and Moore, M.N.  1981.  "Some Practical
Considerations in the Measurement of Pollution Effects on Bivalve
Molluscs, and Some Possible Ecological Consequences," Aquatic
Toxicology, vol 1, pp 159-174.

Bayne, B.L., Moore, M.N., Widdows, J., Livingstone, D.R., and Salkeld,
P.N.  1979.  "Measurement of the Responses of Individuals to
Environmental Stress and Pollution," Philosophical Transactions of the
Royal Society (B), vol 286, pp 563-581.

Bayne, B.L., Widdows, J., and Thompson, R.J.  1976.  "Physiological
integrations," Marine Mussels:  Their Ecology and Physiology, Edited by
Bayne, B.L.  Cambridge University Press, Cambridge, pp 261-291.

Bower, C.E., and Holm-Hansen, T.  1980.  "A Salicylate-Hypochlorate
Method for Determining Ammonia in Seawater," Canadian Journal of
Fisheries and Aquatic Science, vol 37, pp 794-798.

Brown, B.E., and Newell, R.C.  1972.  "The Effect of Copper and Zinc on
the Metabolism of the Mussel Mytilus edulis," Marine Biology, vol 16, pp
108-118.

Conover, R.J.  1966.  "Assimilation of organic matter by zooplankton,"
Limnology and Oceanography, vol 11, pp 338-354.

Crisp, D.J.  1971.  "Energy Flow Measurements," Methods for the Study
of Marine Benthos, IBP Handbook No. 16, Edited by Holme, N.A., and
Mclntyre, A.D.  Blackwell Scientific Publications, Oxford, pp 197-279.

Elliot, J.M., and Davidson, W.  1975.  "Energy Equivalents of Oxygen
Consumption in Animal Energetics," Oecologia, vol 19, pp 195-201.

Engel, D.W., and Fowler, B.A.  1979.  "Copper and Cadmium Induced
Changes in the Metabolism and Structure of Molluscan Gill Tissue,"
Marine Pollution;  Functional Responses, Edited by Vernberg, W.B.,
Thurberg, F.P., Calabrese, A., and Vernberg, F.J.  Academic Press, New
York, pp 239-256.
                                 40

-------
 Gilfillan,  E.S.   1975.   "Decrease  of  Net  Carbon  Flux in  Two  Species  of
 Mussels Caused by Extracts  of  Crude Oil," Marine Biology, vol  29,  pp
 53-57.

 Gilfillan,  E.S.,  Mayo,  D.,  Hanson, S., Donovan,  D.,  and  Jiang,  L.C.
 1976.   "Reduction in Carbon Flux in Mya arenaria Caused  by a Spill of
 No.  6 Fuel  Oil,"  Marine Biology, vol  37,  pp  115-123.

 Guillard, R.R.L.   1975.   "Culture of  Phytoplankton for Feeding  Marine
 Invertebrates," Culture of  Marine Invertebrate Animals,  Edited  by  W.L.
 Smith and M.H.  Chanley,  Plenum Publishing Corp.,  pp  29-60.

 Lake, J., Hoffman, G.,  and  Schiiranel,  S.   1984.   "The  Bioaccumulation of
 Contaminants  from Black Rock Harbor Dredged Material  by  Mussels and
 Polychetes,"  Technical  Report  D-84, prepared by  the U.S.
 Environmental Protection Agency, Narragansett, RI, for the U.S. Army
 Corps of Engineers Waterways Experiment Station,  CE,  Vicksburg, MS.

 Maciolek, J.   1962.   "Limnological Organic Analysis by Quantitative
 Dichromate  Oxidation,"  Fisheries Research Report  60,  pp  1-61.

 Nelson, W.G.   In  Preparation.   "Reproductive cycle of two populations
 of mussels  from Narragansett Bay."

 Nelson, W.G.   In  Preparation.   "Scope for growth  in two  populations  of
 mussels from  Narragansett Bay."

 Phelps, D.K.,  Galloway,  W.B.,  Reynolds, B.H., Nelson, W.G.,  Hoffman,
 G.L., Lake, J.L., Barszcz,  C.,  Thurberg,  F.P., Kraikowski, J.,  and K.
 Jenkins.  1983.   "Evaluation Report:  Use of Caged Mussel Transplants
 for Monitoring Fate  and  Effects of Ocean  Disposal in  the New York Bight
 Apex," Prepared for  the  Office  of Regulations and Standards.  ERLN
 Contribution  No.  586, 36 pp.

 Sastry, A.N.   1975.   "Physiology and Ecology of Reproduction in Marine
 Invertebrates," Physiological  Ecology of  Estuarine Organisms, Edited by
 Vernberg, F.J.  University  of  South Carolina Press, Columbia, South
 Carolina, pp  279-299.

 Scott, D.M.,  and Major,  C.W.   1972.  "The Effect of Copper (II) on
 Survival, Respiration and Heart Rate in the Common Blue Mussel Mytilus
 edulis," Biological  Bulletin, vol 143, pp 679-688.

 Snedecor, G.W., and  Cochran, W.G.  1978.   Statistical Methods,  Iowa
 State University Press,  Iowa, 593 pp.

 Stickle, W.B., Rice, S.D., Villars, C., and Metcalf,  W.  1983.
 "Bioenergetics and Survival of the Marine Mussel, Mytilus edulis,
 During Long-term Exposure to the Water Soluble Fraction of Cook Inlet
 Crude Oil," Abstract from Symposium on Pollution and Physiology of
Marine Organisms.   November  1, 1983, Mystic, Connecticut.
                                  41

-------
Thompson, R.J., and Bayne, B.L.  1974.  "Some Relationships Between
Growth, Metabolism and Food in the Mussel, Mytilus edulis,'' Marine
Biology, vol 27, pp 317-328.

	.  1972.  "Active Metabolism Associated with Feeding in
the Mussel Mytilus edulis," Journal of Experimental Marine Biology and
Ecology, vol 8, pp 191-212.

Warren, C.E., and Davis, G.E.  1967.  "Laboratory Studies on the
Feeding, Bioenergetics and Growth of Fish," The Biological Basis of
Freshwater Fish Production, Edited by Gerking, S.D., Blackwell
Scientific Publications, Oxford, pp 174-219.

Widdows, J., Bayne, B.L., Donkin, P., Livingstone, D.R., Lowe, D.M.,
Moore, M.N., and Salkeld, P.N.  1981.  "Measurement of the Responses of
Mussels to Environmental Stress and Pollutants in Sullom Voe:  A
Baseline Study," Proceedings of the Royal Society of Edinbergh, vol 80
B, pp 323-338.

Widdows, J., Fieth, P., and Worrall, C.M.  1979.  "Relationships
Between Season, Available Food and Feeding Activity in the Common
Mussel Mytilus edulis," Marine Biology, vol 50, pp 195-207.

Widdows, J., Phelps, D.K., and Galloway, W.B.  1981.  "Measurement of
Physiological Condition of Mussels Transplanted Along a Pollution
Gradient in Narragansett Bay," Marine Environmental Research, vol 4, pp
181-194.

Winter, J.E.  1978.  "A Review on the Knowledge of Suspension Feeding
in Lammellibranchiate Bivalves, with Special Reference to Artificial
Aquaculture Systems," Aquaculture, vol 13, pp 1-33.
                                  42

-------
                  APPENDIX A
NO-OBSERVABLE-EFFECT-CONCENTBATION EXPERIMENT

-------
                              Introduction




       1.  As stated in the main text it was determined that in order




to obtain an effect with BRH material it was believed that the mussels




should be exposed to the highest reasonable level of suspended partic-




ulates.  The determination of a reasonable particulate level was




termed the no-observable-effeet-concentration (NOEC) experiment.




Two criteria were used in selecting the NOEC:  (a) maximum exposure




to the suspended sediment, and (b) no significant reduction in SFG.




The null hypothesis was that  there was no particulate level of sus-




pended reference sediment that would have a chronic effect on the




mussels.  As described in Part I of the main text, SFG was measured




under standardized conditions to determine whether some chronic




effect had been incurred during the 28-day exposure.






                         Material and Methods




       2.  To test this hypothesis, reference sediment (REF)  from




Long Island Sound (Lake et al. 1984) was used and mussels were exposed




in groups of twelve each to the following nominal dilution series:




100, 50, 25, 12.5, 6.25,  and 0 mg/£.  Figure 4 shows the mussel




exposure system.  Sediment was delivered to the mixing chamber from




the composite dosing system through a microprocessor-controlled




valve as previously reported by Lake et al. (1984).  For this experi-




ment, the mixing and distribution chambers labeled BRH in Figure 4




contained filtered seawater only, while total suspended particulates




(100 mg/£) were maintained in the REF mixing and distribution




chambers.  The various dilutions were achieved by blending suspended
                                  A2

-------
 particulates  from the REF distribution chamber with the filtered sea-

 water.  Each  exposure chamber contained 12 mussels and received the

 appropriate mixture at  a rate of  100 ml/min.  The duration of the

 experiment was 28 days  with samples of mussels taken on day 28 for

 SFG determinations.  The procedures used for measuring SFG were the

 same as those descibed  in the main text.  The highest particulate

 level that did not produce a negative lasting effect would be chosen

 as the NOEC for the two BRH exposure experiments.

       3.  Measurements of the amount of suspended particulates

 entering the exposure chambers were made daily using a spectrophotom-

 eter, as previously detailed in Part II.  The actual incoming and

 surrounding suspended particulate concentrations are shown in Table Al,

                               Table Al
            Results of  daily monitoring of exposure system
                        in the NOEC experiment.

                               Treatment

                Control   6.25    12.5      25       50        100
Actual
incoming
cone.
(mg/A)

Actual
surrounding
concentration
                 2(0.5)  7(2.3)  12(4.5)  26(6.2)  53(13.8)  106(27.6)
                 7(8.0) 10(7.1)  12(6.5)  15(6.9)   21(9.3)   49(15.6)
Algae             0.094   0.094    0.094    0.094     0.094
(g/mussel/day)
Ref sediment
(g/mussel/day)

Percent Food
0.0   0.075    0.150    0.300     0.600


100    55.6     38.5     23.9      13.5
                                                                 0.094
                                                                 1.200
                                                                   7.3
                                 A3

-------
       4.  The mussels were continuously fed laboratory cultured




T-Iso at a rate of 94 mg  (dry weight) per mussel per day (Table Al).




Conditions and techniques of algal culture were modified after Guillard




(1975).  Guillard's  "f/2" nutrient media was used, except that all




trace metals but  iron were eliminated and the concentration of the




vitamins thiamine and B12 were doubled.






                                Results






Physiological parameters




       5.  Results of the physiological measurements are summarized




in Tables A2 through A5.  They indicate no consistent pattern between




each parameter and the final SFG values (Table A6).  The most probable




reason for this is that some mussels from all treatments, except




100 mg/i, were observed spawning during the physiological measurements




(Table A7).  This point will be discussed more fully in the following




Discussion Section but should be kept in mind as the results in



Tables A2 through A5 are presented.




       6.  The results of the reference sediment experiment are sum-




marized in Tables A2 through A7.  Table A2 lists the weight-specific




mean clearance rates for the mussels from the six treatment levels.



Although there was a wide range in the mean values, there were no




significant differences between any of the treatments.
                                  A4

-------
                                Table A2
           Comparison of mean (standard error) weight-specific
clearance r


Treatment
100
50
Control
12
25
6
ates of ten mussels pei
test with reference

Clearance Rate
(jj/hr)
4.22(0.45)
4.13(0.42)
3.43(0.40)
2.95(0.52)
2.93(0.19)
2.64(0.39)
: treatment in the NOEC
sediment.

Group*
A
A
A
A
A
A
 * Means with  the  same  group  letter  are not  significantly  different.


        7.   The absorption efficiency results  are listed in Table A3.

 Again  there is  a  large variation in the means.   The mussels  from the  25-

 mg/» treatment  were  significantly lower than  the mussels  from the 50-,

 control, and  100-mg/Jl  treatments.
                               Table A3
        The mean  (SE) absorption efficiencies of ten mussels per
treatment

Treatment
•g/l
50
CONTROL
100
12
6
25
in the NOEC reference sediment

Absorption Efficiency
(percent)
76(0.04)
73(0.03)
72(0.03)
66(0.05)
56(0.06)
51(0.07)
experiment .

Group*

A
A
A
A B
A B
B
* Means with the same group letter are not significantly different.


       8.  The mean weight-specific respiration rate for each treatment

is listed in Table A4.  The control group was significantly higher than

all other groups.
                                 A5

-------
                               Table A4
          The mean (SE) weight-specific respiration rates for


Treatment
mg/ft
Control
25
100
50
6
12
the NOEC experiment.

Respiration Rate
(ml 02/hr/g)
1.20(0.07)
0.95(0.05)
0.95(0.05)
0.84(0.06)
0.79(0.03)
0.78(0.02)


Group*

A
B
B
B
B
B
* Means with the same group letter are not significantly different.


       9.  Ammonia excretion rate means for each treatment are listed

in Table A5.  The mussels from the 6-mg/£ treatment had a significantly

higher mean excretion rate than those mussels from the 12-mg/& and

control treatments.  No other differences were observed.

                               Table A5
               Mean (SE) ammonia excretion rates for ten
mussels from each

Treatment
mg/A
6
25
100
50
12
Control
treatment from the

Excretion Rate
(yg NH4-N/hr/g)
29.07(4.64)
21.48(4.59)
20.33(5.91)
15.05(3.12)
11.80(2.19)
7.99(1.99)
NOEC experiment.

Group*

A
A B
A B
A B
B
B
* Means with the same group letter are not significantly different.


Scope for growth index

       10.  The mean SFG values for each treatment in the NOEC experiment

are listed in Table A6.  The 50-, 100-, and 12-mg/A treatments were

statistically similar as were the 12-, 6-, control, and 25-mg/A groups.
                                  A6

-------
 In  addition, the  100-,  12-, and 6-mg/Jl treatments were not statis-

 tically different.  There was a large amount of variability in this

 data set which is reflected in the 95% confidence limits.
                               Table A6
         The mean(SE) weight-specific scope for growth values

Also

Treatment
mg/A
50
100
12
6
Control
25
for each treatment
listed are the 95% <

Scope For Growth
(Joules/hr/g)
1.73(1.85)
-0.96(1.13)
-3.86(1.42)
-7.07(0.98)
-8.48(1.48)
-9.40(1.58)
in the NOEC ex
confidence limi

Group*

A
A B
ABC
B C
C
C
periment.
ts for each mean.

95% Confidence Limits

5.85 to -2.29
1.56 to -3.48
-0.70 to -7.02
-4.89 to -9.25
-6.30 to -10.66
-5.88 to -12.92
* Means with the same group letter are not significantly different.


       11.  Several other measurements were completed on these mussels

and are listed in Table A7.  There was no difference between the mean

length of the mussels in each treatment;  however, the dry weight of

the mussels from the 12- and 50-mg/£ treatments were statistically

higher than those from the 100-mg/fc group.  In addition, several

mussels were observed spawning during and/or after the clearance rate

measurements.  While these numbers are listed in Table A7, there is no

assurance that these were the only mussels to have spawned.  Other

mussels may have spawned either at night or prior to any of the

physiological measurements.
                                  A7

-------
                             Table A7
      Mean (SE) lengths and dry weights of ten mussels from
    each treatment in the NOEC experiment. Also listed are the
     number of mussels observed spawning during physiological
                 measurements from each treatment.

  Treatment     Length(SE) *    Dry Weight(SE)*    Mussels Spawning
    mg/A           (cm)              (g)
12
50
25
6
Control
100
5.33(0.06) A
5.40(0.06) A
5.35(0.04) A
5.33(0.05) A
5.41(0.04) A
5.35(0.05) A
0.66(0.07) A
0.65(0.04) A
0.62(0.03) A B
0.60(0.03) A B
0.55(0.02) A B
0.47(0.04) B
1
1
2
1
8
0
Means with the same group letter are not significantly different.
                                A8

-------
                               Discussion






        12.   The  purpose  this preliminary experiment was to determine




 a suspended  sediment  load  that would have no observeable effect on




 the  SFG of Mytilus edulis.  This concentration was determined to be




 a nominal incoming concentration of 50 mg/&, based on two facts:




 (a)  psuedofeces  were  continuously produced in this treatment, thus




 giving  maximum exposure  to  the suspended sediment; and (b) the mean




 SFG  value was highest for this treatment.  This decision may seem




 arbitrary after  inspection  of  the results of this experiment.  A




 great deal of variability and inconsistency occurred in the data




 from this experiment, most  probably associated with the spawning




 activity during  the measurements.  However, for the purpose of this




 report, which is to test the accuracy and reproducibility of the SFG




 index,  the results are quite important and are included as this appendix.




        13.   The most obvious result of this experiment was the lack




 of a consistent pattern in  the response of the individual physiological




 parameters used to calculate the SFG index, relative to the exposure




 level.  SFG  is an integrative index; however, the data from individual




 physiological measures can  be important to the interpretation of




 results.  In previous studies the effects of particular pollutants




have been documented with M. edulis  (Phelps et  al.  1983,  Stickle




et al.  1983,  Widdows at  al.  1981,  Bayne  et  al.  1979).   In  these




 studies one  of the measured SFG parameters contributed overwhelmingly




 to the observed change in SFG.   The  reference sediment used  in the




 present experiment was from the relatively clean reference site in
                                  A9

-------
Long Island Sound.  The effects observed in the present NOEC experi-




ment, therefore, are probably due to physical action of the suspended




material rather than to a toxic chemical compound.  The results of




the NOEC experiment showed no clear pattern between the individual




physiological paramerers in response to the sediment load.  Three




possible explanations are considered.  First, the exposure system




itself may have introduced variability due to some unidentified




artifact.  Second, the physiological measurements may have introduced ,




some unknown error into the data set.  Third, a factor other than




sediment concentration, i.e., differences in the reproductive condition




of mussels between treatments, may have been influencing the results.




       14.  After inspection of the system monitoring data, it would




appear that the first possibility is not likely.  Table Al shows that




the system was working properly throughout the experiment.




       15.  The second alternative would appear to be unsubstantiated




as well.  For example, the clearance rate data indicated that the




mussels with the highest suspended levels during the experiment (50




and 100 mg/jj,) also had the highest clearance rates (Table A2)




during the SFG measurements.  If this were an artifact from the




experiment, one would expect just the opposite.  Winter (1978) stated




that mussels try to maintain a constant ingestion rate by decreasing




clearance rate with increasing particle concentration.  This same




effect was described by Widdows et al. (1979).  Therefore, the observed




clearance rates do not seem to be due to some factor such as improper



acclimation time before the clearance rate measurement was initiated.
                                 A10

-------
        16.   The absorption efficiency data likewise follow no distinct




pattern.  While variable, the only significant differences were between




the  50- and  25-mg/£ treatments  (Table A3).  Thompson and Bayne (1972)




showed  that  absorption efficiency was inversely proportional to the




suspended particulate load.  If something from the sediment experiment




were affecting this measurement, one would expect differences between




the  highest  and lowest particulate concentrations.  In addition, the




respiration  rates were highest in the control and 100-mg/^ treatments,




which had the greatest difference in particle levels during the test.




        17.   The important conclusion is that no individual physiological




factor was apparently responsible for the SFG differences between




treatments.  In this experiment the factor most closely correlated to




the order of SFG values was the total suspended particulate levels,




and consequently food levels.  The amount of food supplied to each




treatment was the same; however, the particulate levels were different




(Table Al).  In effect, the reference sediment caused a decrease in




the percentage of algae ingested with increasing particle concentration




(Table Al).  This had the effect of supplying more algae to the control




group and proportionally less to each higher particulate level group.



        18.  Differences in food levels between treatments lead to the




third alternative, which offers the most probable explanation for the




observed variability and inconsistencies in the NOEC experiment.




Sastry (1975),  working with another bivalve,  Argopectin irradians,  found



that both an adequate food supply and temperature regime were necessary



to initiate the gametogenic cycle in the bay scallop.   Bayne (1976)
                                All

-------
postulated that the same process was necessary with M. edulis.  In




the present experiment, it is possible that the combination of temper-




ature (constant 15°C) and adequate food supply in the control and




lower particle level treatments may have been sufficient to initiate




the gametogenic cycle.  The higher concentrations may not have had a




sufficient food supply for gametogenesis to occur at the same rate as




the lower concentrations.  The number of mussels spawning during the




SFG measurements may add some credence to this assumption (Table A7).




The control treatment had eight out of ten mussels at least partially




spawn, while the 25-mg/fc treatment had two spawn, and one mussel out




of ten in each of the 6-, 12-, and 50-mg/S- treatments.  No spawning




occurred in the 100-mg/i, treatment mussels.  Bayne et al. (1976) stated




that SFG values are lower during gametogenesis, and this may be a




possible explanation for the variable results.  While this hypothesis is




speculative at the present time, inspection of the reproductive cycle




of the field population from which these mussels were collected may




help to clarify it.



       19.  In another study, Nelson (in prep) followed the gameto-




genic cycle of this population during the past year. , Using sterology




and mantle dry weight as reproductive indices, he found that mussels




from this area exhibited a large spawning peak in late March and a




smaller peak in early December.  This second spawning period coincides




with the collection period for the NOEC experiment.  The mussels




used in this experiment, therefore, were probably at different stages




of the gametogenic cycle.  This fact, in addition to the possible
                                A12

-------
 treatment  food differences mentioned above, may help  to explain  the




 spawning differences observed in this experiment.  The important




 point is that differences in the reproductive condition of experimental




 organisms  can lead to serious problems when the data  are interpreted.




 This is one  factor that must be considered in any experimental design.




     20.   With this problem in mind, the SFG values from this experi-




 ment will  be discussed briefly.  These values can only be compared




 in a relative sense because the physiological measurements were




 completed  under standardized conditions.  A mussel with an SFG value




 of zero is, by definition, at its maintenance ration.  Inspection of




 the 95 percent confidence limits about the means indicate that the




 50-, 100-, and 12-mg/Jl treatments were around that maintenance ration.




 The other  three treatments were below this ration level for this test




 only.  During the physiological measurements, cultured algae were




 supplied at approximately 0.5 mg/£ of seawater.  This ration was




chosen based on the metabolic rates of mussels collected at this




 time of the year in the field (Nelson in prep).  Because this




test was completed in less than 24 hr, it is believed that any sub-




maintenance ration did not adversely affect the animals.  In a rela-




tive sense, therefore, it is believed that the statistical differences




observed between means are valid.  Long-term holding under these




conditions would not be recommended.  However, in the exposure system,




the food levels were considerably higher.




     21.  The final SFG values (Table A6) indicate that the mussels in




the 50-, 100-,  and 12-mg/fc treatments were not different statistically.
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Of these three, only the 50-mg/A treatment did not overlap any other




group.  In light of this fact and the previous information concerning




pseudofecal production, a nominal incoming concentration of 50 mg/& was




chosen as the NOEC for the BRH experiments.




     22.  One problem encountered during the NOEC test was the repro-




ductive condition of different groups of mussels.  This problem was




not encountered during the BRH experiment.  Differences in reproductive




condition can cause complications when interpreting SFG data measured




at different times of the year.  One possible solution would be to




use juvenile mussels in place of adults.  The use of adult mussels




to measure the effects of pollutants, including SFG, is well documented.




With the proper experimental design a hypothesis could be tested to




determine if the same results could be achieved using juvenile mussels




instead of adults.  This would alleviate one source of variability




and possibly make the comparison of SFG results more straightforward.
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